In applied protein chemistry, it is a common problem how to formulate or present a biologically active peptide or protein to the relevant site of activity, typically an organic target, such as a nucleic acid, a protein, a complex of proteins, or a complex of a protein(s) and/or lipids and/or carbohydrates and/or a nucleic acid(s). The simplest solution is simply to provide an aqueous solution of the biologically active peptide or protein. Many applications do however require some further means to achieve the desired goal. For instance, the peptides/proteins may be associated with a lipid mixture or chemically immobilized to a support structure.
Applications for peptides/proteins immobilized to a support structure include preparative and analytical separation procedures, such as bioprocesses, chromatography, cell capture and culture, active filters, and diagnostics. Structures based on extracellular matrix proteins, e.g. collagen, are disclosed in EP 704 532 and EP 985 732.
It has also been suggested to use spider silk proteins in a supporting structure. Spider silks are nature's high-performance polymers, obtaining extraordinary toughness and extensibility due to a combination of strength and elasticity. Spiders have up to seven different glands which produce a variety of silk types with different mechanical properties and functions. Dragline silk, produced by the major ampullate gland, is the toughest fiber. It consists of two main polypeptides, mostly referred to as major ampullate spidroin (MaSp) 1 and 2, but e.g. as ADF-3 and ADF-4 in Araneus diadematus. These proteins have molecular masses in the range of 200-720 kDa. Spider dragline silk proteins, or MaSps, have a tripartite composition; a non-repetitive N-terminal domain, a central repetitive region comprised of many iterated poly-Ala/Gly segments, and a non-repetitive C-terminal domain. It is generally believed that the repetitive region forms intermolecular contacts in the silk fibers, while the precise functions of the terminal domains are less clear. It is also believed that in association with fiber formation, the repetitive region undergoes a structural conversion from random coil and α-helical conformation to β-sheet structure. The C-terminal region of spidroins is generally conserved between spider species and silk types.
WO 07/078239 and Stark, M. et al., Biomacromolecules 8: 1695-1701, (2007) disclose a miniature spider silk protein consisting of a repetitive fragment with a high content of Ala and Gly and a C-terminal fragment of a protein, as well as soluble fusion proteins comprising the spider silk protein. Fibers of the spider silk protein are obtained spontaneously upon liberation of the spider silk protein from its fusion partner.
Rising, A. et al., CMLS 68(2): 169-184 (2010) reviews advances in the production of spider silk proteins.
US 2009/0263430 discloses chemical coupling of the enzyme β-galactosidase to films of a miniature spider silk protein. However, chemical coupling may require conditions which are unfavourable for protein stability and/or function. Proteins containing multiple repeats of a segment derived from the repetitive region of spider silk proteins have been designed to include a RGD cell-binding segment (Bini, E et al., Biomacromolecules 7:3139-3145 (2006)) and/or a R5 peptide (Wong Po Foo, C et al., Proc Natl Acad Sci 103 (25): 9428-9433 (2006)) or other protein segments involved in mineralization (Huang, J et al., Biomaterials 28: 2358-2367 (2007); WO 2006/076711). In these prior art documents, films are formed by solubilizing the fusion proteins in the denaturing organic solvent hexafluoroisopropanol (HFIP) and drying.
US 2005/261479 A1 discloses a method of for purification of recombinant silk proteins having an affinity tag, involving magnetic affinity separation of individual silk proteins from complex mixtures without formation of silk protein fibers or other polymer structures.
Known supporting structures and associated techniques have certain drawbacks with regard to e.g. economy, efficiency, stability, regenerating capacity, bioactivity and biocompatibility.